PRCP and PREP belong to prolyl peptidase family. Phylogenetic analysis shows that PRCP and PREP contain highly similar amino acid sequences, and have a similar enzyme function. They can cleave proline-containing substrates such as neuropeptide angiotensin II/III (AngII/III) and α-melanocyte stimulating hormone (α-MSH). PREP additionally cleaves nerve vasopressin (neurotensin) and gastrin-releasing hormone and other neuropeptides. These neuropeptides can activate G protein-coupled receptors (GPCR) and regulate the function of the receptor tyrosine kinase signaling pathway through the G protein-coupled receptors (Garcia-Horsman et al, (2007) Neutopeptides 41: 1-24; Rosenblum J S et al “(2003) Current Opinion in Chemical Biology, 7:496-504; Skidgel et al, (1998) Immunological Reviews, 161: 129-41. Rozengurt E et al, (2010) Clin Cancer Res; 16: 2505-11). Current research indicates that PRCP plays a role in obesity (Shariat-Madar B et al, (2010) Diabetes Metab Syndr Obes, 3: 67-78). Our previous study teaches that PRCP regulates breast cancer cell proliferation, autophagy, and resistance to the drug tamoxifen (Duan L et al, (2011) JBC, 286:2864-2876). PREP is also associated with amnesia, depression and Alzheimer's disease (Rosenblum J S et al, (2003) Current Opinion in Chemical Biology 7:496-504). Cell growth and proliferation are regulated by a number of different factors, including the availability of nutrients, growth factors (such as insulin and insulin-like growth factor, etc.) as well as the availability of the energy state of the cell, etc. PI3K/AKT/mTOR provide signal pathway integration of these factors to control cell growth and proliferation (Manning et al, (2007) Cell 129: 1261-1274; Engelman et al, (2006) Nat Rev Genet 7:606-619). Aberrant activation of PI3K/AKT/mTOR signaling pathway is considered to be the most common feature of all cancers (Engelman, J A. (2009) Nature Reviews/Cancer 9:550-562).
PI3K/AKT/mTOR signaling pathway is activated by RTKs (receptor tyrosine kinases), including the insulin receptor (IR), insulin-like growth factor receptor (IGF-1R), platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR). RTKs can activate PI3K directly or indirectly through insulin receptor substrate (IRS) that interacts with PI3K ρ85 subunit and further activates PI3K p110 catalytic subunits (Markman et al., (2009) Ann Oncol. 21 (4): 683-91).
PI3K is an intracellular phosphatidylinositol kinase. There are three types of PI3K. Class I PI3Ks are mostly cytosolic, are heterodimers comprised of a p110 catalytic subunit and an adaptor/regulatory subunit, and are further divided into two subclasses: Class IA PI3Ks consist of a p110 catalytic subunit that associates with an SH2 domain-containing subunit p85, and is activated by the majority of tyrosine kinase-coupled transmembrane receptors; class IB PI3K consists of a p101 regulatory subunit that associates with p110γ catalytic subunit, and is activated by heterotrimeric GPCR. (Katso et al. (2001) Annu. Rev. Cell Dev. Biol. 17:615). Class II PI3Ks consist of three isoforms, as discussed herein. Class III PI3Ks utilize only phosphatidylinositol as a substrate, and play an essential role in protein trafficking through the lysosome. (Volinia, et al. (1995) EMBO J. 14:3339).
Class IA PI3K activity is suppressed in cytosol by p85 regulatory subunits that form heterodimers with the p110 catalytic subunit. IRS proteins (including IRS-1, IRS-1, IRS-3, IRS-4) are insulin receptor (IR) and insulin-like growth factor receptor (IGF-1R) adapter proteins. IR/IGF1R activates PI3K by regulating IRS protein tyrosine phosphorylation and subsequent interaction with PI3K p85 subunit. Many cancer tissues overexpress insulin receptor substrate IRS-1, while transgenic overexpression of IRS-1 or IRS-2 in mice caused breast cancer tumorigenesis and metastasis (Metz, et al, (2011) Clin Cancer Res 17: 206-211; Bergmann et al, (1996) Biochem Biophys Res Commun 220: 886-890; Dearth et al, (2006) Mol Cell Biol 26: 9302-9314). Tyrosine phosphorylation of IRS proteins is regulated by IR/IGF-IR and other RTKs such as EGFR and ErbB3 which activate IRS proteins. IRS proteins are also regulated by a number of serine/threonine kinases (for example, PKC, mTOR, S6K and ERK) that phosphorylate IRS proteins on serine leading to protein degradation and inhibition of IRS proteins (Copps et al (2012). Diabetologia. 55(10): 2565-2582). Degradation of insulin receptor substrates by certain drugs results in cell death in melanoma (Reuveni et al (2013) Cancer Res 73: 4383-4394). IRS proteins phosphorylated on tyrosine interact with the SH2 domain of p85 subunit resulting in recruitment of PI3K to membrane and release of the inhibitory effect of p85 leading to activation of PI3K. PI3Ks are enzymes that phosphorylate the 3-hydroxyl position of the inositol ring of phosphoinositides (“PIs”). Activated PI3K generates phosphatidylinositol 3-phosphate (PI3P) that serves as a secondary messenger in growth signaling pathways, influencing cellular events including cell survival, migration, motility, and proliferation; oncogenic transformation; tissue neovascularization; and intracellular protein trafficking. PI3P activates the PI3K-dependent protein kinase-1 (PDK1), which in turn activates the kinase AKT. AKT phosphorylates downstream target molecules to promote cell proliferation, survival and neovascularization. (Cantley et al. (1999) PNAS 96:4240) mTOR is an important signaling molecule downstream of the PI3K/AKT pathway (Grunwald et al. (2002) Cancer Res. 62: 6141; Stolovich et al. (2002) Mol Cell Biol. 22: 8101). AKT-mediated phosphorylation inhibits the GAP activity of TSC1/TSC2 toward the Rheb GTPase, leading to Rheb activation. Rheb binds directly to mTOR, a process that is regulated by amino acids. Both amino acids and Rheb activation are required for mTOR activation. mTOR downstream effector molecules include p70 S6 ribosomal protein kinase (S6K) and eukaryotic initiation factor binding inhibitory protein (4E-BP1). After the activation mTOR phosphorylates and activates the catalytic activity S6K1. mTOR also catalyzes phosphorylation of 4E-BP1 and inactivates it, resulting in initiation of protein translation and cell cycle progression (Kozma et al, (2002) Bioessays 24: 65). More importantly, mTOR exerts a negative feedback on activation of PI3K/AKT by suppressing expression and activation of IRS proteins. Inhibition of mTOR by rapamycin relieves the negative inhibition leading to activation of PI3K AKT (Shi et al (2005) Mol Cancer Ther 2005; 4(10): 1533).
PI3K/AKT/mTOR signaling pathway inhibition is considered a promising cancer treatment (Engelman, J A, (2009) Nature Reviews: Cancer 9:551). mTOR antagonist rapamycin is the first signaling target in the PI3K/AKT/mTOR pathway for anti-cancer treatment (Courtney et al, (2010) J Clin Oncol 28: 1075-1083; Vivanco et al, (2002) Nat Rev Cancer 2:489-501). Unfortunately, rapamycin lifts the negative feedback inhibition of IRS proteins, leading to the activation of PI3K and AKT. Patients treated by rapamycin show increased AKT phosphorylation in tumors, leading to failure of tumor treatment (Easton et al, (2006) Cancer Cell. 9 (3):153-5) Therefore, there is a need to develop means to effectively inhibit the PI3K/AKT/mTOR signaling pathway, in particular to prevent the feedback activation of IRS proteins upon inhibition of mTOR.